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Method for the in Vitro Diagnosis and Prognosis of Demyelinating Diseases, and for the Development of Drugs Against Demyelinating Diseases

a demyelinating disease and in vitro diagnosis technology, applied in the field of demyelinating disease in vitro diagnosis and prognosis, and the development of demyelinating disease drugs, can solve the problems of receptor overexcitation, severe axonal damage, and no medication has been generated to delay or stop the progression of the neurodegenerative phas

Inactive Publication Date: 2008-05-15
PROGENIKA BIOPHARMA SA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013]The main object of the present invention is the development of an in vitro method for detecting the presence of demyelinating diseases, for determining the stage or severity of said diseases in the individual, or for monitoring the effect of the therapy administered to an individual presenting said diseases.

Problems solved by technology

However, no medication has been generated which delays or stops the progression of the neurodegenerative phase of the disease which takes a course with progressive neurological degeneration, and which is characterized by the occurrence of severe demyelinating lesions in the white substance with massive oligodendrocyte loss, atrophy and severe axonal damage.
Decrease of the efficacy of glutamic acid transport may cause the receptor overexcitation and trigger neuronal death.
Oligodendrocytes are the cells responsible for central nervous system myelinization, and their death causes severe alterations in nervous communication.
Likewise, it has been observed that overactivation of glutamatergic receptors causes oligodendroglial death.
However, the cellular mechanisms leading to either process, and the molecules intervening, are not well known.

Method used

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  • Method for the in Vitro Diagnosis and Prognosis of Demyelinating Diseases, and for the Development of Drugs Against Demyelinating Diseases
  • Method for the in Vitro Diagnosis and Prognosis of Demyelinating Diseases, and for the Development of Drugs Against Demyelinating Diseases
  • Method for the in Vitro Diagnosis and Prognosis of Demyelinating Diseases, and for the Development of Drugs Against Demyelinating Diseases

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Experimental program
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Effect test

example 1

Differential Analysis of dusp6 Gene Expression in Oligodendrocyte Samples Using the Microarrays Rat Genome U34 DNA Arrays

1.1. Materials and Methods

[0088]Microarrays. The microarrays GeneChip Test 3 (Affymetrix, Santa Clara) were used which allow testing the quality of the RNA prior to the expression analysis with GeneChip Rat Genome U34 set (Affymetrix, Santa Clara), representing 3322 complete gene sequences. The dusp6 gene is represented in the microarray by probe set U42627_at of Affymetrix, which are sense oligonucleotides of 25 nucleotides in length designed on the basis of GeneBank U42627 sequence (Table 1).

TABLE 1Description of probes corresponding to U42627_at probe set.ConsecutivePosition of probeorder ofin mRNA sequenceprobesProbe sequence (5′-3′)of gene1SEQ ID NO: 515572SEQ ID NO: 615633SEQ ID NO: 716234SEQ ID NO: 817135SEQ ID NO: 917256SEQ ID NO: 1017737SEQ ID NO: 1118278SEQ ID NO: 1218459SEQ ID NO: 13185710SEQ ID NO: 14186311SEQ ID NO: 15188112SEQ ID NO: 16188713SEQ ID N...

example 2

Differential Analysis of DUSP6 Protein Expression in Oligodendrocyte Cultures, Using the Real Time Quantitative RT-PCR Technique

2.1. Materials and Methods.

[0114]The method used consists of reverse transcription of mRNA to cDNA and its subsequent amplification into a LightCycler equipment (Roche), using SYBR Green for the detection of the amplified product. Real time quantification is carried out, and it allows calculating the relative expression of the sequence in different samples in the linear amplification phase of the reaction.

[0115]Samples: Primary oligodendrocyte culture samples treated with AMPA were analyzed. The samples came from cultures different from those analyzed with DNA microarrays. 4 cultures were carried out:

[0116]control: untreated cultured oligodendrocytes

[0117]ampa 2: oligodendrocytes treated for 2 minutes with 10 μM AMPA and collected after treatment

[0118]ampa 10: oligodendrocytes treated for 10 minutes with 10 μM AMPA collected after treatment

[0119]ampa 15: ol...

example 3

Cell Death Inhibition in Oligodendrocytes Treated with AMPA after Incubation with Antisense Oligonucleotides Against the dusp6 Gene

3.1. Materials and Methods.

[0142]To evaluate if dusp6 expression blocking inhibits cell death of oligodendrocytes treated with AMPA, antisense oligonucleotides (ODNs) capable of blocking said gene expression were used. The working model used in this assay is a model based on the cell death of oligodendrocytes, a typical model for study the neurodegenerative phase of said demyelinating diseases.

[0143]Oligonucleotide design was carried out based on the secondary structure of the RNA, trying to prevent sequences presenting a high internal hybridization percentage, and with the aid of AO predict (http: / / www.cgb.ki.se / AOpredict / ) computer software. To improve efficacy and capability to degrade RNA and to reduce side effects, last-generation oligonucleotides were designed, presenting two types of modifications; 2′-O-Methyl groups in the 6 first and last bases,...

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Abstract

The present invention refers to an in vitro method for detecting the presence of demyelinating diseases in an individual, for determining the stage or severity of said diseases in the individual, or for monitoring the effect of the therapy administered to an individual suffering said diseases; to the search, identification, development and evaluation of efficacy of compounds for therapy of said diseases for the purpose of developing new drugs; as well as to agents inhibiting DUSP6 protein expression and / or activity, and / or the effects of this expression. The methods and agents of the invention are preferably applied to multiple sclerosis.

Description

FIELD OF THE INVENTION[0001]The present invention refers to an in vitro method for detecting the presence of demyelinating diseases in an individual, for detecting the stage or severity of said diseases in the individual, or for monitoring the effect of the therapy administered to an individual presenting said diseases; to the search, identification, development and evaluation of efficacy of compounds for therapy of said diseases for the purpose of developing new medicaments; as well as to agents inhibiting the expression and / or activity of the DUSP6 protein, and / or to the effects of this expression. The methods and agents of the invention are preferably applied to multiple sclerosis.BACKGROUND OF THE INVENTION[0002]Demyelinating diseases are those in which the main pathogenic process causes the destruction of the myelin sheath, which is necessary for the integrity of central nervous system cells. The main demyelinating diseases are: multiple sclerosis, Devic's syndrome, Baló diseas...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C07H21/04C12N9/16C12Q1/42G01N33/50G01N33/564G01N33/573
CPCC12N9/16C12Q1/42G01N2800/285G01N33/564G01N33/5088
Inventor ILLARRAMENDI, AINARA VALLEJOMARTINEZ, ANTONIO MARTINEZLOPEZ DE CASTRO, USUE ARIZORTIZ DE MENDIBIL, LOURDES OSABASANCHEZ-VALLEJO, CORINA JUNQUERACRUZ, SIMON SANTAGARCIA DE GALDEANO, PEDRO ESCUDEROGARAY, JORGE OCHOABUELA, LAUREANO SIMONALMAU, CARLOS MATUTEALFONSO, ELENA ALBERDIGOMEZ, MARIA VICTORIA SANCHEZBILBAO, GASKON IBARRETXEGALNARES, ESTIBALIZ ETXEBARRIA
Owner PROGENIKA BIOPHARMA SA